Articles of the scientific periodical and patent literature are cited throughout the specification. Each such article is hereby incorporated by reference in its entirety by such citation.
Gram negative bacterial endotoxin is a widespread contaminant of materials employed in biomedical arts, both research and clinical. All pharmaceutical solutions used for parenteral or injection administration must be certified as to endotoxin levels. Also, media used for tissue culture applications are typically tested for the presence of endotoxin. It is of great importance, therefore, to be able to produce a reliably reproducible test for the presence of endotoxin in a sample. Also, it would be an advance in the art to provide materials and methods for removing Gram negative bacterial endotoxins from formulations intended for pharmaceutical or other uses requiring preparations substantially free of endotoxin.
Factor C is a component of the Limulus amoebocyte lysate (LAL) assay that is presently the standard method for testing for endotoxin. Factor C is a serine protease proenzyme that is the first protein in a cascade that leads to coagulation of the LAL upon contact with endotoxin. Factor C is the protein that actually binds to the endotoxin; upon binding of endotoxin, the serine protease activity of Factor C becomes activated. The activated Factor C initiates a chain of proteolytic steps culminating in formation of a solid clot of protein from the LAL. The time for forming the LAL clot and the degree of gelation is indicative of the amount of endotoxin in a sample (Ho, B., Kim, J. C., and Ding, J. L., 1993, Biochem. Mol. Biol. Intl. 29, 687-694). However, this gelation assay is subjective, and variable in its sensitivity of detection of endotoxin due to batch-to-batch variation in the amoebocyte lysate preparation. It is therefore desirable to express recombinant Factor C which could be standardized for use in an assay for endotoxin detection.
There are other endotoxin tests presently known, for example, the United States Pharmacopeia (USP) rabbit pyrogen assay (Tomasulo, P A., Levin, J. Murphy, P A. & Winkelstein, J A. 1977. J. Lab. Clin. Met. 89, 308-315). However, the USP rabbit pyrogen test is not only time consuming, it is also expensive and often gives variable results in detecting endotoxin in pharmaceuticals and parentals (Muller-Calgon, H., pp. 343-356; "Endotoxins and their Detection with the LAL test," S. Watson et al., ed., c. 1982 by Alan R. Liss, New York N.Y.).
Enzyme-linked-immunosorbent assay (ELISA) has been developed for testing evoked secretion of interleukin-6 from monocytic cell lines in response to pyrogen or endotoxin (Taktak, Y. S., Selkirk, S., Bristow, A F., Carpenter, A., Ball, C., Rafferty, B., & Poole, S.,J. Pharm. Pharmacol. 43, 578-582 (1991)). However, this test is probably more suited for research purposes.
One other form of endotoxin detection involves its localization in tissues of experimental animals during induced endotoxemia. An immunohistochemical method utilizes native Factor C (purified from T. tridentatus) to specifically bind the endotoxin. The Factor C-endotoxin complex is then revealed by labelled anti-Factor C antibody (Takeuchi, M. et al., Pathol. Res. Pract. 190(12): 1123-1133 (1994); Nakao, A. et al., Eur. Surg. Res. 27(4): 216-221 (1995)). This study indicates another utility for Factor C. It is therefore within the embodiment of this application to obtain truncated recombinant constructs (e.g., pHILD2/CrFC21/EE containing the 5' end of CrFC cDNA insert flanked by EcoR1 sites).
cDNAs encoding Factor C proteins from Carcinoscorpius rotundicauda have been previously described (U.S. Ser. No. 08/296,014 and J. L. Ding, A. A. Navas III and B. Ho, Mol. Marine Biol. and Biotech. 4:90-103 (1995)). Recombinant Factor C from Carcinoscorpius rotundicauda (rCrFC) has been produced in vitro by coupled transcription/translation systems (U.S. Ser. No. 08/296,014 and S. D. Roopashree et al. Biochem. and Mol. Biol. Int'l. 35:841-849 (1995)). However, the present invention resides partly in the development of in vivo systems, especially using yeasts as a host cell, for efficient production of rCrFC by expression of cloned DNA.
Also, the protection of rCrFC from activation and subsequent self-proteolysis by binding of endotoxin which may be present in solutions used in isolation of the protein is described in U.S. Ser. No. 08/296,014. Basically, dimethylsulfoxide (Me.sub.2 SO, DMSO) is added to solutions which are used during the purification process. Even greater protection of the rCrFC is achieved by also adding an agent effective for chelating divalent metal ions to the purification solutions.
As a means to circumvent difficulties in determining endotoxin in plasma due to endogenous interfering factors, a chromogenic LAL assay was modified to include a specific step to adsorb the plasma endotoxin using immobilized histidine. Endotoxin in samples was separated from interfering factors by chromatography through immobilized histidine in which endotoxin was specifically adsorbed and, subsequently quantified by fluorimetric LAL assay (Nawata, M., Minobe, S., Hase, M., Watanabe, T., Sato, T. & Tosa, T., J. Chromatogr., 597: 415-424 (1992); Minobe, S., Nawata, M., Shigemori, N. & Watanabe, T., Eur. J. Clin. Chem. Clin. Biochem., 32(10): 797-803 (1994). However, this method of endotoxin adsorption is limited only to small volumes of 0.5-1 ml, and there is no report on the use or feasibility of this method for removing endotoxin from large preparations. It has so far only been reported as an improved method of endotoxin assay, albeit one limited by infeasibility of chromogenic assay of the endotoxin bound to the column.
More recently, Qiagen (US) has marketed a `Qiagen` kit for purification of endotoxin-free plasmids (See, Qiagen News Issue No. 1, 1996). This may involve a specific (proprietary) reagent that removes endotoxin from the plasmid preparation.